The present invention is directed to methods of manufacture of nanoparticles and approaches for such manufacture. In certain preferred embodiments of the invention, the nanoparticles may be used to encode information and thereby serve as molecular (or cellular) tags, labels and substrates.
The present invention relates to methods of manufacture of segmented particles and assemblies of differentiable particles (which may or may not be segmented). Without a doubt, there has been a paradigm change in what is traditionally defined as bioanalytical chemistry. A major focus of these new technologies is to generate what could be called xe2x80x9cincreased per volume information contentxe2x80x9d. This term encompasses several approaches, from reduction in the volume of sample required to carry out an assay, to highly parallel measurements (xe2x80x9cmultiplexingxe2x80x9d), such as those involving immobilized molecular arrays, to incorporation of second (or third) information channels, such as in 2-D gel electrophoresis or CE-electrospray MS/MS.
Unfortunately, many of these seemingly revolutionary technologies are limited by a reliance on relatively pedestrian materials, methods, and analyses. For example, development of DNA microarrays (xe2x80x9cgene chipsxe2x80x9d) for analysis of gene expression and genotyping by Affymetrix, Incyte and similar companies has generated the wherewithal to immobilize up to 20,000 different fragments or full-length pieces of DNA in a spatially-defined 1-cm2 array. At the same time, however, the use of these chips in all cases requires hybridization of DNA in solution to DNA immobilized on a planar surface, which is marked both by a decrease in the efficiency of hybridization (especially for cDNA) and a far greater degree of non-specific binding. It is unclear whether these problems can be completely overcome. Moreover, there is a general sense of disillusionment both about the cost of acquiring external technology and the lead-time required to develop DNA arraying internally.
A second example of how groundbreaking can be slowed by inferior tools is in pharmaceutical discovery by combinatorial chemistry. At the moment, solution phase, 5-10 xcexcm diameter latex beads are used extensively as sites for molecular immobilization. Exploiting the widely adopted xe2x80x9csplit and poolxe2x80x9d strategy, libraries of upwards of 100,000 compounds can be simply and rapidly generated. As a result, the bottleneck in drug discovery has shifted from synthesis to screening, and equally importantly, to compound identification, (i.e., which compound is on which bead?). Current approaches to the latter comprise xe2x80x9cbead encodingxe2x80x9d, whereby each synthetic step applied to a bead is recorded by parallel addition of an organic xe2x80x9ccodexe2x80x9d molecule; reading the code allows the identity of the drug lead on the bead to be identified. Unfortunately, the xe2x80x9ccode readingxe2x80x9d protocols are far from optimal: in most every strategy, the code molecule must be cleaved from the bead and separately analyzed by HPLC, mass spectrometry or other methods. In other words, there is at present no way to identify potentially interesting drug candidates by direct, rapid interrogation of the beads on which they reside, even though there are numerous screening protocols in which such a capability would be desirable.
Two alternative technologies with potential relevance both to combinatorial chemistry and genetic analysis involve xe2x80x9cself-encoded beadsxe2x80x9d, in which a spectrally identifiable bead substitutes for a spatially defined position. In the approach pioneered by walt and co-workers, beads are chemically modified with a ratio of fluorescent dyes intended to uniquely identify the beads, which are then further modified with a unique chemistry (e.g. a different antibody or enzyme). The beads are then randomly dispersed on an etched fiber array so that one bead associates with each fiber. The identity of the bead is ascertained by its fluorescence readout, and the analyte is detected by fluorescence readout at the same fiber in a different spectral region. The seminal paper (Michael et al., Anal. Chem. 70, 1242-1248 (1998)) on this topic points out that with 6 different dyes (15 combinations of pairs) and with 10 different ratios of dyes, 150 xe2x80x9cunique optical signaturesxe2x80x9d could be generated, each representing a different bead xe2x80x9cflavorxe2x80x9d. A very similar strategy is described by workers at Luminex, who combine flavored beads ready for chemical modification (100 commercially available) with a flow cytometry-like analysis. (See, e.g., McDade et al., Med. Rev. Diag. Indust. 19, 75-82 (1997)). Once again, the particle flavor is determined by fluorescence, and once the biochemistry is put onto the bead, any spectrally distinct fluorescence generated due to the presence of analyte can be read out. Note that as currently configured, it is necessary to use one color of laser to interrogate the particle flavor, and another, separate laser to excite the bioassay fluorophores.
A more significant concern with self-encoded latex beads is the limitations imposed by the wide bandwidth associated with molecular fluorescence. If the frequency space of molecular fluorescence is used both for encoding and for bioassay analysis, it is hard to imagine how, for example, up to 20,000 different flavors could be generated. This problem might be alleviated somewhat by the use of combinations of glass-coated quantum dots, which exhibit narrower fluorescence bandwidths. (See, e.g. Bruchez et al., Science, 281, 2013-2016 (1998)). However, these xe2x80x9cdesignerxe2x80x9d nanoparticles arc quite difficult to prepare, and at the moment, there exist more types of fluorophores than (published) quantum dots. If, however, it were possible to generate very large numbers of intrinsically-differentiablc particles by some means, then particle-based bioanalysis would become exceptionally attractive, insofar as a single technology platform could then be considered for the multiple high-information content research areas; including combinatorial chemistry, genomics, and proteomics (via multiplexed immunoassays).
Previous work has originally taught how metal can be deposited into the pores of a metallized membrane to make an array of metal nanoparticles embedded in the host. Their focus was on the optical and/or electrochemical properties of these materials. A similar technique was used to make segmented cylindrical magnetic nanoparticles in a host membrane, where the composition of the particles was varied along the length. In no case, however, have freestanding, rod-shaped nanoparticles with variable compositions along their length been prepared. Indeed, xe2x80x9cfreestandingxe2x80x9d rod-shaped metal nanoparticlcs of a single composition, in which the length is at least one micron, have never been reported. Likewise, freestanding rod-shaped metal nanoparticles not embedded or otherwise contained within such host materials have never been reported. See, Martin et a., Adv. Materials 11 1021-25 (1999).
Rod-shaped nanoparticles have been prepared whose composition is varied along the length of the rod. These particles are referred to as nanoparticles or nanobar codes, though in reality some or all dimensions may be in the micron size range. The present invention is directed to methods of manufacture of such nanoparticles.
The present invention includes methods of manufacture of freestanding particles comprising a plurality of segment, wherein the particle length is from 10 nm to 50 xcexcm and particle width is from 5 nm to 50 xcexcm. The segments of the particles of the present invention may be comprised of any material. Included among the possible materials are a metal, any metal chalcogenide, a metal oxide, a metal sulfide, a metal selenide, a metal telluride, a metal alloy, a icital nitride, a metal phosphide, a metal antimonide, a semiconductor, a semi-metal, any organic compound or material, any inorganic compound or material, a particulate layer of material or a composite material. The segments of the particles of the present invention may be comprised of polymeric materials, crystalline or non-crystalline materials, amorphous materials or glasses. In certain preferred embodiments of the invention, the particles are xe2x80x9cfunctionalizedxe2x80x9d (e.g., have their surface coated with IgG antibody). Commonly, such functionalization may be attached on selected or all segments, on the body or one or both lips of the particle. The functionalization may actually coat segments or the entire particle. Such functionalization may include organic compounds, such as an antibody, an antibody fragment or an oligonucleotide, inorganic compounds, and combinations thereof. Such functionalization may also be a detectable tag or comprise a species that will bind a detectable tag.
Also included within the present invention are methods of manufacture of an assembly or collection of particles comprising a plurality of types of particles, wherein each particle is from 20 nm to 50 xcexcm in length and is comprised of a plurality of segments, and wherein the types of particles are differentiable. In the preferred embodiments, the particle types are differentiable based on differences in the length, width or shape of the particles and/or the number, composition, length or pattern of said segments. In other embodiments, the particles arc differentiable based on the nature of their functionalization or physical properties (e.g., as measured by mass spectrometry or light scattering).
The present invention includes the manufacture of nanobar codes by the electrochemical deposition of metals inside a template wherein the process is improved, separately and collectively, by i) electroplating in an ultrasonication bath; and ii) controlling the temperature of the deposition environment, preferably by using a recirculating temperature bath.
Also included within the scope of the invention are methods for the simultaneous or parallel manufacture of a plurality of different types of nanobar codes. According to one such method, a plurality of templates are held in a common solution chamber and electrochemical deposition is accomplished by controlling deposition at each membrane by applying current selectively to predetermined electrodes associated with each such membrane.
Also included within this invention is an apparatus for the manufacture of nanobar codes comprising: a plating solution cell, a defined pore size template, means for applying a current to cause electrochemical deposition of a metal into said template, means for agitation of the plating solution, such as an ultrasonic transducer, and temperature control means.
Also included within this invention is an apparatus for the simultaneous manufacture of a plurality of different types of nanobar codes. In one embodiment, such apparatus comprises: a solution chamber, a plurality of templates, means for selectively applying a current to each of said templates, and control means for operating said apparatus.